Use of polymer d-lactic acid (pdla) or equivalents thereof to inhibit growth of cancer cells and diagnose cancers

ABSTRACT

The invention provides method for sequestering or trapping L-lactate in or near a tumor cell comprising contacting an isolated polymer of D-lactic acid (PDLA) or an equivalent, derivative or analog thereof with the tumor cell so that the PDLA binds L-lactate in or near the cell and thereby sequestering or trapping L-lactate in or near the tumor cells.

This patent application is a divisional application of U.S. Ser. No.13/360,567, filed Jan. 27, 2012, which claims the benefit of the filingdate of U.S. Ser. No. 61/462,103, filed Jan. 28, 2011, the contents ofall of which are herein incorporated by reference in their entiretiesinto the present patent application.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

In 1931, Otto Warburg was awarded the Nobel Prize in medicine for hispioneering work demonstrating glycolysis as the primary anaerobicglucose metabolism within cancer cells (Warburg O W F, Negelein E., JGen Physiol. 1927; 8:519-530). Unfortunately, to date, a universaltherapy has not emerged from his work. Since that time, it has becomeapparent that not all cancer cells utilize glycolysis to produce ATP,but some utilize oxidative phosphorylation (Krebs cycle) to generateenergy (Seyfried T N, Shelton L M. Nutr Metab (Lond). 2010; 7:7). Morespecifically, those cells in the interior of a tumor where the oxygentension is lowest and the milieu more acidotic tend to utilizeglycolysis while those cells in the periphery where oxygen tension ishigher tend to utilize oxidative phosphorylation (Vaupel P. Semin RadiatOncol. July 2004; 14(3):198-206), Glycolysis generates protons that needto be transported out of the cell to avoid acid build up. Many of thehydrogen ions that are transported out of the cell are accompanied bylactate to maintain electrical neutrality. There are other mechanismsexclusive of lactate that generate anions to buffer the acid productionof glycolysis. These include metabolism of pyruvate to bicarbonate byhydration of CO2 catalyzed by various carbonic anhydrases andmonocarboxylases (Halestrap A P. UCSD-Nature Molecule Pages. 29 Oct.2009:1-20). Decreasing the intracellular lactate concentration may causecell death from unchecked acidosis. The intracellular pHs of theinterior and exterior tumor cells are similar but the extracellularfluid surrounding the inner cells is orders of magnitude more acidic(Gerweck L E, et al. Mol Cancer Ther. May 2006; 5(5):1275-1279). It hasbeen shown that a lactate shuttle exists between inner and outer cellswhere lactate is converted to pyruvate in the outer cells and thenmetabolized by oxidative phosphorylation to produce ATP to sustaincancer cells (Brooks G A. J. Physiol. Dec. 1, 2009; 587(Pt23):5591-5600).

The explanation of which tumor cells utilize glycolysis is more complexthan oxygen availability. Rapid rates of energy production require theexpedient but inefficient fermentation of glucose if there areinsufficient or functionally abnormal mitochondria to process glucosethrough oxidative phosphorylation (Seyfried T N, Shelton L M. Nutr Metab(Lond). 2010; 7:7). In addition to the metabolic effects of lactate inglycolysis, lactate has also been shown to contribute to tumor cellinvasion and increased cell motility (Gatenby R A, et al. Cancer Res.May 15, 2006; 66(10):5216-5223).

More recent investigations have tried to separate lactate productionfrom the hypoxic effects on tumor growth by using genetic markers. Thesestudies suggest lactic acidosis may be independent of oxygen tension andmay be associated with a more favorable clinical outcome (Chen J L, etal. PLoS Genet, December 2008; 4(12):e1000293). If this concept is true,then inactivation or trapping of lactate could worsen clinical outcome.

One major problem associated with present medications that target tumormetabolism is that they also target normal cells. The compositions ofthe invention solves the problem in the art. The compositions of theinvention are tumoricidal and has the advantage that it is less harmfulto cells having mitochondria with the capacity to shuttle pyruvate thanthe subset of cancer cells that exclusively utilize glycolysis.

SUMMARY OF THE INVENTION

The invention provides methods for sequestering or trapping L-lactate inor near a tumor cell comprising contacting an isolated polymer ofD-lactic acid (PDLA) or an equivalent, derivative or analog thereof withthe tumor cell so that the PDLA binds L-lactate in or near the cell andthereby sequestering or trapping L-lactate in or near the tumor cells.

The invention also provides methods for determining intracellular orextracellular L-lactate levels comprising exposing a PDLA or anequivalent, derivative or analog thereof to or near a cell so as to bindL-lactic acid in or near the cell and thereby forming a complex, whereinthe PDLA is labeled, and detecting the complex.

The invention further provides compositions comprising isolated PDLA oran equivalent, derivative or analog thereof having a molecular weight(MW) of less than 500 Daltons, wherein the composition is substantiallyfree of PDLA or an equivalent, derivative or analog thereof having a MWgreater than 500 Daltons.

The invention also provides compositions comprising isolated PDLA or anequivalent, derivative or analog thereof having fewer than 12 monomersof D-lactic acid, wherein the composition is substantially free of PDLAor an equivalent, derivative or analog thereof having more than 12monomers of D-lactic acid.

The invention also provides kits for sequestering, depleting, measuring,or detecting L-lactic acid in solution, in cell culture, in serum, in acell, or in an animal comprising isolated PDLA or an equivalent,derivative or analog thereof either unmodified or modified.

The invention also provides methods for treating a cancer comprisingadministering to a subject an isolated PDLA or an equivalent, derivativeor analog thereof under sufficient conditions so as to inhibit tumorcells in the subject thereby treating the subject having a cancer.

The invention further provides methods for preventing cancer bysequestering or trapping L-lactate comprising administering to a subjectan isolated PDLA or an equivalent, derivative or analog thereof in anamount effective to sequester or trap L-lactate so as to prevent cancerin the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows l-lactate (white) binds (yellow) to an isolated PDLAoligomer (green). PDLA oligomer shown is an isolated polymer of fourD-lactic acid arising from condensation polymerization involving thehydroxyl group next to the carboxylic acid group of one D-lactic acidresidue with the carboxyl group of an adjacent D-lactic acid residueleading to formation of an ester bond and loss of a water molecule. Forthe PDLA shown, there are three ester bonds and three water moleculeslost during condensation polymerization. The orientation of the PDLAshown, where n, the number of D-lactic acid residues, equals 4, has theunreacted hydroxyl group at the left end of the polymer and theunreacted carboxylic acid group at the right end. Although a linear formof PDLA is shown in the figure, PDLA can exist in a circular form withno free hydroxyl or carboxylic acid group but with an ester bond betweenadjacent D-lactic acid residues.

FIG. 2-1 through 2-3. Interaction of D-lactic acid or L-lactic acid withpoly-D-lactic acid. The plots show absorption (uV; Y axis), andretention time (min; X axis). Frame 1, D-lactic acid alone; Frame 2,Poly-D-lactic acid alone; Frame 3, D-lactic acid plus poly-D-lacticacid; Frame 4, L-lactic acid alone; Frame 5, poly-D-lactic acid alone;Frame 6, L-lactic acid plus poly-D-lactic acid. Addition ofpoly-D-lactic acid (2) to D-lactic acid (1) does not modify thechromatogram of the combination (3). Addition of poly-D-lactic acid (5)to L-lactic acid (4) modifies the chromatogram of the combination (6).This indicates that poly-D-lactic acid binds L-lactic acid but notD-lactic acid.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety.

As used herein, the term “comprising” when placed before the recitationof steps in a method means that the method encompasses one or more stepsthat are additional to those expressly recited, and that the additionalone or more steps may be performed before, between, and/or after therecited steps. For example, a method comprising steps a, b, and cencompasses a method of steps a, b, x, and c, a method of steps a, b, c,and x, as well as a method of steps x, a, b, and c. Furthermore, theterm “comprising” when placed before the recitation of steps in a methoddoes not (although it may) require sequential performance of the listedsteps, unless the content clearly dictates otherwise. For example, amethod comprising steps a, b, and c encompasses, for example, a methodof performing steps in the order of steps a, c, and b, the order ofsteps c, b, and a, and the order of steps c, a, and b. Unless otherwiseindicated, all numbers expressing quantities of ingredients, propertiessuch as molecular weight, reaction conditions, and so forth as usedherein, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters herein are approximations that may vary dependingupon the desired properties sought to be obtained by the presentinvention. At the very least, and without limiting the application ofthe doctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parametersdescribing the broad scope of the invention are approximations, thenumerical values in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains standarddeviations that necessarily result from the errors found in thenumerical value's testing measurements.

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal. Mammals include but are not limited to, humans,murines, simians, felines, canines, equines, bovines, porcines, ovines,caprines, rabbits, mammalian farm animals, mammalian sport animals, andmammalian pets. In many embodiments, the subject will be humans.

As used herein, the term “isolated” or “purified” in reference to PDLAdoes not require absolute purity.

PDLA compositions of the invention may be produced by using principlesof chemistry or biochemistry.

As used herein, “inhibition” or “treatment” of a cancer means to providean intervention that ameliorates the symptoms of the cancer, reduces theseverity of the cancer, alters the course of disease progression, and/orameliorates or cures the basic disease problem. For example, treatmentof a cancer may be accomplished by sequestering or trapping L-lactate.Inhibition or treatment may be partial or total.

The most effective mode of administration and dosage regimen for thecompositions of the invention depends upon the location, extent, or typeof the disease being treated, the severity and course of the medicaldisorder, the subject's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions of the invention should be titrated to the individualsubject and/or by the specific medical condition or disease.

By way of example, the interrelationship of dosages for animals ofvarious sizes and species and humans based on mg/m² of surface area iswell known. Adjustments in the dosage regimen may be made to optimizesuppression or modulation of the cancer e.g., doses may be divided andadministered on a daily basis or weekly or biweekly or monthly basis orthe dose reduced proportionally depending upon the situation (e.g.,several divided doses may be administered daily or proportionallyreduced depending on the specific therapeutic situation).

As is well known, the dose of the composition of the invention requiredto achieve an appropriate clinical outcome may be further reduced withschedule optimization.

As used herein, polymer D-lactic acid (PDLA) is also known aspoly((R)-2-Hydroxypropanoic acid), (R)-2-Hydroxypropanoic acidhomopolymer, poly((R)-2-Hydroxypropionic acid),poly((R)-ethylidenelactic acid), poly((R)-1-Hydroxyethanecarboxylicacid) or D-lactic acid homopolymer.

Compositions of the Invention

The present invention provides a novel composition comprising isolatedPDLA or an equivalent, derivative or analog thereof having a molecularweight (MW) of 500 Daltons or less, wherein the composition issubstantially free of PDLA or an equivalent, derivative or analogthereof having a MW greater than 500 Daltons. PDLA or an equivalent,derivative or analog thereof may be in the form of a linear polymer withfree hydroxyl group next to an ester group at one end and a freecarboxylic acid group at the other end or may be in the form of acircular polymer in which one D-lactic acid residue is joined by anester bond to an adjacent D-lactic acid residue with no free hydroxyl orcarboxylic acid group.

In one embodiment, the composition comprises isolated PDLA or anequivalent, derivative or analog thereof having fewer than 12 monomersof D-lactic acid, wherein the composition is substantially free of PDLAor an equivalent, derivative or analog thereof having more than 12monomers of D-lactic acid.

In another embodiment, the composition contains isolated PDLA or anequivalent, derivative or analog thereof having 7 monomers of D-lacticacid or fewer. In yet another embodiment, the composition that containsisolated PDLA or an equivalent, derivative or analog thereof that have 7monomers of D-lactic acid or fewer is substantially free of PDLA or anequivalent, derivative or analog thereof having more than 7 monomers ofD-lactic acid.

In a further embodiment, the composition contains isolated PDLA or anequivalent, derivative or analog thereof that have between 2 to 4monomers of D-lactic acid. In yet another embodiment, the compositionthat contains isolated PDLA or an equivalent, derivative or analogthereof that have between 2 to 4 monomers of D-lactic acid issubstantially free of PDLA or an equivalent, derivative or analogthereof having fewer than 2 monomers of D-lactic acid. Additionally, inanother embodiment, the composition that contains isolated PDLA or anequivalent, derivative or analog thereof that have between 2 to 4monomers of D-lactic acid is substantially free of PDLA or anequivalent, derivative or analog thereof having more than 4 monomers ofD-lactic acid. Moreover, an additional embodiment provides a compositionthat contains isolated PDLA or an equivalent, derivative or analogthereof that have between 2 to 4 monomers of D-lactic acid and issubstantially free of PDLA or an equivalent, derivative or analogthereof having (1) fewer than 2 monomers of D-lactic acid and (2) morethan 4 monomers of D-lactic acid.

Polymers of d-lactic acid (PDLA) or an equivalent, derivative or analogthereof may form a stereocomplex (FIG. 1) with l-lactate in solution, invitro, or in vivo, as in Experiments #1-3, producing lactate deficiencyin tumor cells, such as human chronic lymphocytic leukemia (CLL) cellsin Experiment #3. Those cancer cells that utilize transport of lactateto maintain electrical neutrality may cease to multiply or die becauseof lactate trapping, and those cancer cells that benefit fromutilization of extracellular lactate may be impaired. Intracellulartrapping of lactate may produce a different physiology than inhibitionof LDH because the cell loses the option of shuttling pyruvate to analternative pathway to produce an anion.

Conjugated with stains or fluorescent probes, isolated PDLA or anequivalent, derivative or analog thereof of the invention may be anagent for the diagnosis of tissue lactate and possibly celldifferentiation in biopsy specimens. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme.

Additionally, the PDLA or an equivalent, derivative or analog thereof ofthe invention may be conjugated to cytotoxic agents. Examples ofcytotoxic agents include, but are not limited to ricin, ricin A-chain,doxorubicin, daunorubicin, taxol, ethiduim bromide, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin(PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin,gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin,crotin, calicheamicin, sapaonaria officinalis inhibitor, maytansinoids,and glucocorticoid and other chemotherapeutic agents, as well asradioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Additional cytotoxic agents include, but are not limited toDichloroacetate (DCA), 2-deoxyglucose (2-DG) and Lonidamine.

DCA

DCA is a pyruvate dehydrogenase activator, inhibiting the activity ofpyruvate dehydrogenase kinase (PDK), and thus promoting the conversionof pyruvate to Acetyl-CoA (Madhok B M, et al. Br J Cancer. Jun. 8, 2010;102(12):1746-1752). Treatment with DCA shifts the fate of pyruvate fromglycolysis to mitochondrial oxidative phosphorylation which is likelyresponsible for its tumoricidal activity. DCA is an inhibitor of allfour isoenymes of PDK (Madhok B M, et al. Br J Cancer. Jun. 8 2010;102(12):1746-1752). DCA has been used for the treatment of congenitaland acquired lactic acidosis with the most significant long term sideeffect of reversible peripheral neuropathy but sedation and elevation ofhepatic transaminases have also been reported. The dose required toinhibit colorectal cancer cells in vivo is estimated to be 5-10 timesthat used in treatment of lactic acidosis (Madhok B M, et al. Br JCancer. Jun. 8, 2010; 102(12):1746-1752; Papandreou I, et al. Int JCancer. Oct. 18, 2010). Potential serum therapeutic doses could bebetween 20 and 50 mmole/L (Madhok B M, et al. Br J Cancer. Jun. 8, 2010;102(12):1746-1752). In a number of studies, DCA has been shown to be atumoricidal agent and has reduced lactate levels in growth media in adose-dependent manner (Michelakis E D, et al. Sci Transl Med. May 12,2010; 2(31):31ra34). Some favorable results have been reported in fivepatients suffering from glioblastoma multiforme where DCA was used incombination with surgery, temozolomide and radiation (Papandreou I, etal. Int J Cancer. Oct. 18, 2010).

2-DG

2-DG is a glucose analog that is a competitive inhibitor of glucose. Itis actively transported, into cells and phosphorylated into 2-DCphosphate (2-DC-P) by hexokinase (Michelakis E D, et al. Sci Transl Med.May 12, 2010; 2(31):31ra34). Further metabolism of 2-DC-P is notpossible and thus glycolysis is limited. 2-DC also has additionaleffects on protein glycosylation separate from its effects on glycolysisand it may enhance radionuclide cytotoxicity (Dwarakanath B S. J CancerRes Ther. September 2009; 5 Suppl 1:527-31; Shrivastava V, et al. JCancer Res Ther. April-June 2006; 2(2):57-64; Raiser M, et al. Proc NatlAcad Sci USA. Nov. 18, 2008; 105(46):17807-17811). In vivo studies (inmice) have shown that 2-DG is synergistic with adriamycin and paclitaxelin transplanted human osteosarcoma and non-small cell lung cancer(Pelicano H, et al., Oncogene. Aug. 7, 2006; 25(34):4633-4646; PrasannaV K, et al. J Cancer Res Ther. September 2009; 5 Suppl 1:S44-47).Combination chemotherapy with 2-DG has been trialed in patients withglioblastoma (Prasanna V K, et al. J Cancer Res Ther. September 2009; 5Suppl 1:S44-47). Because 2-DG is an analog of glucose it producesactions and side effects in cancer and non-cancer cells. 2-DG may haveproconvulsant actions (Gasior M, et al. Epilepsia. August 2010;51(8):1385-1394).

Lonidamine

Lonidamine, a derivative of indazole-3-carboxylic acid is an inhibitorof oxygen consumption and suppresses glycolysis in cancer cells probablythrough hexokinase suppression (Pelicano H, et al. Oncogene. Aug. 7,2006; 25(34):4633-4646). Its action is non-selective, but synergisticwith other chemotherapeutic agents and it has been used in clinicaltrials for breast, ovarian, lung and glial cancers (Pelicano H, et al.,Oncogene. Aug. 7, 2006; 25(34):4633-4646). The major complicationobserved in more than a majority of patients was myalgia that requiredlowering the dose (Evans W K, et al. Oncology. 1984; 41 Suppl 1:69-77).

Other metabolic chemotherapeutic agents that may be conjugated to theisolated PDLA of the compositions of the invention may include:3-broinopyruvate, an inhibitor of hexokinase, oxythiamine, an inhibitorof transketolase and pyruvate dehydrogenase, and 6-aminonicotinamide, aninhibitor of the pentose phosphate pathway (Pelicano H, et al. Oncogene.Aug. 7, 2006; 25(34):4633-4646; Gatenby R A, Gillies R J. Int J BiochemCell Biol. 2007; 39(7-8):1358-1366).

Compositions herein may comprise one or more PDLA oligomers providedherein. The compositions are, in one embodiment, formulated intosuitable pharmaceutical preparations such as solutions, suspensions,tablets, dispersible tablets, pills, capsules, powders, sustainedrelease formulations or elixirs, for oral administration or in sterilesolutions or suspensions for parenteral administration, as well astransdermal patch preparation and dry powder inhalers. In oneembodiment, the compositions described above are formulated intopharmaceutical compositions using techniques and procedures well knownin the art (see, e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof is (are) mixed with asuitable pharmaceutical carrier. The PDLA of the compositions of theinvention may be derivatized prior to formulation, as described above.The concentrations of the PDLA oligomers in the compositions areeffective for delivery of an amount, upon administration, that treats,prevents, or ameliorates one or more of the symptoms of diseases ordisorders to be treated.

In one embodiment, the compositions are formulated for single dosageadministration.

The composition may be included in a pharmaceutically acceptable carrierin an amount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compositions in in vitro systems described herein, and thenextrapolated therefrom for dosages for humans.

The concentration of active compound in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theactive compound, the physicochemical characteristics of the compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompositions and derivatives thereof may be, in one embodiment,formulated and administered in unit-dosage forms or multiple-dosageforms. Unit-dose forms as used herein refers to physically discreteunits suitable for human and animal subjects and packaged individuallyas is known in the art. Each unit-dose contains a predetermined quantityof the therapeutically active composition sufficient to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier, vehicle or diluent. Examples of unit-dose formsinclude ampoules and syringes and individually packaged tablets orcapsules. Unit-dose forms may be administered in fractions or multiplesthereof. A multiple-dose form is a plurality of identical unit-dosageforms packaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecomposition as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

A. Compositions for Oral Administration

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms may be tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

1. Solid Compositions for Oral Administration

In certain embodiments, the formulations are solid dosage forms, in oneembodiment, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcompounds of a similar nature: a binder; a lubricant; a diluent; aglidant; a disintegrating agent; a coloring agent; a sweetening agent; aflavoring agent; a wetting agent; an emetic coating; and a film coating.Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, molasses,polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The composition of the invention may further include agents that protectit from the acidic environment of the stomach. For example, thecomposition can be formulated in an enteric coating that maintains itsintegrity in the stomach and releases the active compound in theintestine. The composition may also be formulated in combination with anantacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is a compound or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

2. Liquid Compositions for Oral Administration

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is in oneembodiment encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are known. For a liquid dosageform, the solution, e.g., for example, in a polyethylene glycol, may bediluted with a sufficient quantity of a pharmaceutically acceptableliquid carrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

B. Injectables, Solutions and Emulsions

Parenteral administration, in one embodiment characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained is also contemplated herein.Briefly, a composition provided herein may be dispersed e.g., in a solidinner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate,plasticized or unplasticized polyvinylchloride, plasticized nylon,plasticized polyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinyl alcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinyl chloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The compound diffuses through the outer polymeric membrane in a releaserate controlling step. The percentage of active compound contained insuch parenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN™ 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intra-arterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

C. Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

D. Targeted Formulations

The compounds provided herein, or pharmaceutically acceptablederivatives thereof, may also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art.

E. Combination Therapy

In another embodiment, the compositions may be administered incombination, or sequentially, with another therapeutic agent. Such othertherapeutic agents include those known for treatment, prevention, oramelioration of one or more symptoms of amyloidosis andneurodegenerative diseases and disorders.

Methods of the Invention

The invention additionally provides method for sequestering or trappingL-lactate in or near a tumor cell. In one embodiment, the methodcomprises contacting an isolated PDLA or a variant, derivative or analogthereof or any of the compositions of the invention described hereinwith the tumor cell so that the PDLA binds L-lactate in or near the celland thereby sequestering or trapping L-lactate in or near the tumorcells.

The invention also provides methods for inhibiting tumor cells bysequestering or trapping L-lactate in the tumor cell by contacting anisolated PDLA or a variant, derivative or analog thereof (or any of thecompositions of the invention described herewith) with the tumor cell sothat the PDLA binds L-lactate in or near the cell. Additionally, theinvention provides methods for killing or inhibiting cancer bysequestering or trapping L-lactate in the tumor cell by contacting anisolated PDLA or a variant, derivative or analog thereof (or anycompositions of the invention described herein) with the tumor cell sothat the PDLA binds L-lactate in or near the cell. The PDLA or avariant, derivative or analog thereof (or any of the compositionsdescribed herein) may be modified to be resistant to esterases.

In accordance with the practice of the invention, the PDLA or a variant,derivative or analog thereof (including in any of the compositions ofthe invention described herein) may be conjugated to a soluble molecule.Examples of soluble molecules include but are not limited to a dextran,polyethylene glycol, and dendrimer. Additionally, the PDLA or a variant,derivative or analog thereof (including in any of the compositions ofthe invention described herein) may be conjugated with a therapeutic,detectable, or imaging agent.

Further, the invention provides methods for determining intracellular orextracellular L-lactate levels. In one embodiment, the method comprisesexposing PDLA or a variant, derivative or analog thereof (or anycompositions of the invention described herein) to or near a cell so asto bind L-lactic acid in or near the cell and thereby forming a complex.The PDLA or a variant, derivative or analog thereof (or any of thecompositions of the invention described herein) may be labeled, and sothat the complex is detected.

In accordance with the practice of the invention, the PDLA or a variant,derivative or analog thereof (including in any of the compositions ofthe invention described herein) may be labeled with a radioactiveisotope, a fluorescent dye or a calorimetric agent.

In an embodiment of the invention, the method of detection may beeffected by fluorescence resonance energy transfer (FRET). In thisexample, the fluorescent dye may be chemical compounds, such asfluorescein and rhodamine or Cy3™ and Cy5™, or a fluorescent proteinsuch as the green fluorescent protein (GFP) and mutants or derivativesthereof, such as ECFP or EYFP.

In an embodiment of the invention, the method of detection may beeffected by detection of a pronounced fluorescence upon binding ofL-lactic acid by PDLA or a variant, derivative or analog thereof(including in any of the compositions of the invention described herein)labeled with a fluorescent dye (e.g., Oregon Green™ 488-X, 6-FAM™, TET™,Cy3™, Rhodamine Red®-X, TAMRA™, ROX™, Bodipy 630/650™-X, Bodipy650/665™-X, and Cy5™) and a fluorescent dye quencher (e.g., Dabcyl,BHQ™-1, BHQ™-2, and Iowa Black™ FQ/RQ) or a pair of fluorescent dyesthat can interact in fluorescence resonance energy transfer. Forexample, presence of one fluorescent dye on one end of PDLA or avariant, derivative or analog thereof (including in any of thecompositions of the invention described herein) and a quencher or itsFRET pair on the other end produces a notable difference in the emissionfluorescence spectrum (either wavelength or amplitude) in the absence orpresence of L-lactic acid. A change in the emission fluorescencespectrum is produced upon binding to L-lactic acid as the distance andorientation of the two labeled dyes are changed.

The invention further provides methods for treating a cancer comprisingadministering to a subject an isolated PDLA or a variant, derivative oranalog thereof (or any of the compositions of the invention describedherein) under sufficient conditions so as to inhibit tumor cells.

Examples of cancers include but are not limited to colorectal cancer,osteosarcoma, non-small cell lung cancer, breast cancer, ovarian cancer,glial cancer, solid tumors, metastatic tumor, acute lymphoblasticleukemia, acute myelogenous leukemia, adrenocortical carcinoma, Kaposisarcoma, lymphoma, anal cancer, astrocytomas, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancer, brain tumor, breast cancer,bronchial tumor, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic myeloproliferative disorders, coloncancer, colorectal cancers, ductal carcinoma in situ, endometrialcancer, esophageal cancer, eye cancer, intraocular, retinoblastoma,metastatic melanoma, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumors,glioblastoma, glioma, hairy cell leukemia, head and neck cancer,hepatocellular carcinoma, hepatoma, Hodgkin lymphoma, hypopharyngealcancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oralcavity cancer, liver cancer, lobular carcinoma in situ, lung cancer,non-small cell lung cancer, small cell lung cancer, lymphoma,AIDS-related lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma, cutaneousT-cell lymphoma, melanoma, squamous neck cancer, mouth cancer, multiplemyeloma, myelodysplastic syndromes, myelodysplastic/myeloproliferativeneoplasms, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic carcinoma, papillarycarcinomas, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, pineal parenchymal tumors, pineoblastoma, pituitarytumor, pleuropulmonary blastoma, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell cancer, salivarygland cancer, sarcoma, Ewing sarcoma, soft tissue sarcoma, squamous cellcarcinoma, Sezary syndrome, skin cancer, Merkel cell carcinoma,testicular cancer, throat cancer, thymoma, thymic carcinoma, thyroidcancer, urethral cancer, endometrial cancer, uterine cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia,and Wilms tumor.

The invention also provides for methods for preventing cancer bysequestering or trapping L-lactate in an effective amount comprisingadministering to a subject an isolated PDLA or a variant, derivative oranalog thereof (or any of the compositions of the invention describedherein) under sufficient conditions so as to prevent the cancer.

KITS

According to another aspect of the invention, kits are provided. Forexample, the invention provides kits for sequestering, depleting,measuring, or detecting L-lactic acid in solution, in cell culture, inserum, in a cell, or in an animal comprising isolated PDLA or a variant,derivative or analog thereof (or any of the compositions of theinvention described herein) of the invention either unmodified ormodified. For example, isolated PDLA may be used free in solution orconjugated to a solid support to deplete lactic acid produced duringfermentation, such as in beer and wine manufacturing process. Kitsaccording to the invention include package(s) comprising compounds orcompositions of the invention.

The phrase “package” means any vessel containing compounds orcompositions presented herein. In preferred embodiments, the package canbe a box or wrapping. Packaging materials for use in packagingpharmaceutical products are well known to those of skill in the art.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.

The kit can also contain items that are not contained within the packagebut may be attached to the outside of the package, for example,pipettes.

Kits may optionally contain instructions for administering thecompositions of the invention to a subject having a condition in need oftreatment. Kits may also comprise instructions for approved uses ofcompounds herein by regulatory agencies, such as the United States Foodand Drug Administration. Kits may optionally contain labeling or productinserts for the present compositions. The package(s) and/or any productinsert(s) may themselves be approved by regulatory agencies. The kitscan include compositions in the solid phase or in a liquid phase (suchas buffers provided) in a package. The kits also can include buffers forpreparing solutions for conducting the methods, and pipettes fortransferring liquids from one container to another.

The kit may optionally also contain one or more other compounds for usein combination therapies as described herein. In certain embodiments,the package(s) is a container for intravenous administration. In stillother embodiments, compositions are provided in a polymeric matrix or inthe form of a liposome.

The following examples are provided to further illustrate aspects of theinvention. These examples are non-limiting and should not be construedas limiting any aspect of the invention.

EXAMPLES

PDLA Interferes with L-Lactate Measurement (Methods and Results)

Experiment #1

Forty mg of d-lactic acid (Sigma-Aldrich, St. Louis, Mo.; catalog no.L0625) was polymerized in a microwave oven according to the method ofPandey and Aswath (J Biomater Sci Polym Ed. 2009; 20(1):33-48). Briefly,d-lactic acid was transferred to 2 ml Reacti-Vials (Pierce Chemical Co.,Rockford, Ill.) and the polymerization was carried out vented in a GESpacemaker Model JVM 1650WH05 (Serial #900524B; manufactured April 2006)with an RF output frequency of 2450 MHz in the absence of any addedsolvent or catalyst. Polymerization was carried out starting at Power 1setting on the microwave and weighing at 30 second intervals. Powersetting was increased stepwise until about Power 4 which resulted in asample weight of 20 mg corresponding to 50% weight loss. Weight loss wasfrom water during polymer esterification and dehydration. During thecourse of the microwave process, d-lactic acid crystals undergo a statechange from a solid to a liquid and then to a gel at the end of theprocess. Similar procedure was used to produce PLLA with l-lactic acid.

The polymerization process may be monitored by taking samples during thecourse of the microwave treatment and analyzed by Fourier transforminfrared (FT-IR) spectroscopy, proton nuclear magnetic resonance(1H-NMR) spectroscopy and/or gel-permeation chromatograph (GPC). Thefinal synthesized PDLA or PLLA may be further purified or isolated toobtain the desired molecular weight or number of lactic acid residues inthe oligomer by gel-permeation chromatography, as described in Pandeyand Aswath (J Biomater Sci Polym Ed. 2009; 20(1):33-48).

In normal saline, at a temperature of 37 degrees centigrade, a controlsolution of 1 ml of l-lactic acid (50 mg/L) (Sigma) and separately 1 mlof an experimental solution of 1 ml of 50 mg/L of l-lactic acid with 20mg of PDLA were incubated for 2 hours. Measurement of l-lactate withL-Lactate Accuvin Test strips (Accuvin LLC, Napa, Calif.), usingstereospecific lactate dehydrogenase and a tetrazolium color indicator,measured less l-lactate (interference) in the experimental sample asevidenced by colorimetric determination. Experimental sample vs. controlmeasured 10 mg/L vs. 50 mg/L of l-lactate. Other relevant tests usingsimilar methodology and D-Lactate Accuvin Test strips are as follows:

1. Interference when Polymer L-Lactate (PLLA) (100 mg) was added to a 1ml solution of 800 mg/L d-lactic acid.

2. No interference when 50 mg/L l-lactic acid solution was added to a 60mg/L d-lactic acid solution. (Table 1.)

TABLE 1 Interference reactions of PDLA and l- lactate & PLLA andd-lactate in saline l-lactate d-lactate test strip test strip l-lacticacid (0.05 mg) 50 mg/L l-lactic acid (0.05 mg) + PDLA (20 mg) 10 mg/Ld-lactic acid (0.8 mg) 800 mg/L d-lactic (0.8 mg) acid + PLLA (100 mg)not detected d-lactic acid (0.05 mg) + l-lactic 150 mg/L  acid (0.06 mg)

Conclusion of experiment #1: PDLA in normal saline solution decreasesthe measurement of l-lactate in normal saline solution and PLLA innormal saline solution decreases the measurement of d-lactate in normalsaline solution.

Experiment #2

After approval from the director of the clinical laboratory and thechairman of the human studies committee of the Durham Veterans AffairsMedical Center and after vigorous exercise, 18 ml of blood was drawnfrom a human subject. The blood was collected in three vials eachcontaining sodium fluoride/potassium oxalate. Tubes 1 and 3 werecentrifuged at 3400 rpm for 15 minutes and the serum was frozen. To tube2 was added and mixed 0.5 ml of a 1 ml normal saline solution containinga mixture of PDLA d-lactate oligomers with an initial weight of 100 mgof d-lactic acid that had been microwave polymerized with loss of 30 mgof water. Tube 2 was then centrifuged at 3400 rpm and the serum wasfrozen. Tubes 1-3 containing serum were frozen at −5 degrees centigradefor 12 hours and then defrosted in an incubator at 37 degrees centigradefor 3 hours prior to assay. The tubes were then placed on ice andprocessed in the laboratory.

Serum was processed using the Siemens Dimension Vista System Flexreagent cartridge at the Durham Veterans Affairs Medical Center.Reference values of l-lactate are in the range of 0.4-2.0 mmol/L (Table2.)

TABLE 2 PDLA decreases measurement of l-lactate in blood Tube l-lactateconc. PDLA l-lactate conc, with dilution 1 9.1 mmol/L 0 9.1 mmol/L 2 7.0mmol/L 0.5 ml 7.58 mmol/L  3 8.6 mmol/L 0 8.6 mmol/L

Conclusion of experiment #2: PDLA of unknown molecular weight oligomersreduces the measurement of l-lactate in blood samples without additionof catalyst or heat.

Proposed Reaction

l-lactate+PDLA=PDLA˜l-lactate complex+PDLA+l-lactate

Experiment #3

To determine the ability of PDLA and PLLA to mediate cytotoxicity formalignant cells, freshly isolated human chronic lymphocytic leukemia(CLL) cells were cultured with the agents for 72 hours at 37° C. in 5%carbon dioxide/95% air using SFM™ tissue culture medium (Invitrogen,Carlsbad, Calif.). The cells were cultured in triplicate with eight2-fold dilutions of the agents (from 2.000 to 0.016 mg/mL). Cytotoxicitywas determined with an assay using the tetrazolium-based compound MTS(Promega, Madison, Wis.) (Levesque M C, et al. Leukemia. February 2003;17(2):442-450).

Conclusion of experiment #3: The studies demonstrated cytotoxicity forthe CLL cells, with the mean effective dose for 50% cytotoxic effect of1.30 mg/mL for PDLA (1.93 and 0.66 mg/mL in patients A and B), and 2.14mg/mL for PLLA (2.65 and 1.63 mg/mL in patients A and B). Thus, the PDLAwas effective at a lower concentration than PLLA.

Stereocomplex Model of PDLA and L-Lactate

The beta helix strand formed by PDLA oligomers could template forl-lactate because the lactate anion is attracted to the carbonyl of theester which carries a partial positive charge because of the inductiveeffects of the adjacent oxygen molecules. Hydrogen bonding could existbetween the OH group of l-lactate and the ester oxygen of oligomers ofPDLA. With these attractions, the methyl groups of PLDA and lactate arefavorably oriented with minimal steric effects. This complex which hasbeen more specifically named a homo-stereocomplex is probably distinctfrom the stereo PLDA˜PLLA complex. The minimum size oligomer of PDLAthat would be needed to complex intracellular l-lactate may be less than500 Daltons. (FIG. 1)

Discussion of Experimental Section

The experimental section supports that PDLA oligomers form astereocomplex with l-lactate. The reaction occurs spontaneously in bothnormal saline and plasma (delta G negative and K_(equilibrium) greaterthan 1). Separation of the polymer to oligomers less than 500 Daltons(231, 308, 385 or 462 corresponding to 3×, 4×, 5×, or 6× d-lactatemonomers respectively) may be the ideal weights for biologic applicationassuming that rapid plasma hydrolysis does not occur in vivo andmultiple binding sites exist for l-lactate to complex with PDLAoligomers. (Table 3.)

TABLE 3 PDLA oligomer weights and hydrogen bond acceptors and donors #of d-lactate Hydrogen Hydrogen monomers Molecular weight bond acceptorsbond donors 2 154 5 0-2 3 231 7 0-2 4 308 9 0-2 5 385 11 0-2 6 462 130-2

Estimation of Three Compartment (Blood, Extracellular Tumor,Intracellular Tumor) PDLA Concentrations

The size, charge and lipophilic properties of the PDLA oligomers willmostly determine the likelihood that it will be available in theextracellular space to complex l-lactate. Using control values ofpH_(e)=6.77 (extracellular pH) and pHp=7.4 (plasma pH), assuming noactive transport, the ion distribution of PDLA with a pKa 3.86 would beapproximately (Gerweck L E, et al. Mol Cancer Ther. May 2006;5(5):1275-1279):

pHp=3.86+log [PDLA_(p) ⁻]/[PDLA_(p) ] { capillary }  pH_(e)=3.86 + log[PDLA_(e) ⁻]/[PDLA_(e)] 3.54=log [PDLA_(p) ⁻]/[PDLA_(p) ] { capillary } 2.91= log [PDLA_(e) ⁻]/[PDLA_(e)] Antilog of equations 3470=[PDLA_(p)⁻]/[PDLA_(p)] { capillary }  2042=[PDLA_(e) ⁻]/[PDLA_(e)]      [PDLA_(p)]~ [PDLA_(e) ] (Assuming equilibrium across thecapillary) [PDLA_(p) ⁻ ]/ [PDLA⁻ _(e)]=3470/2042=1.70

Thus the approximate plasma concentration of the anion oligomer would be1.70× that of the extracellular concentration.

The size, charge and lipophilic properties of the oligomer will mostlydetermine the likelihood that it will be available in the cytosol tocomplex l-lactate. However, the acid milieu of the extracellularcompartment surrounding the tumor will aid in intracellular ion trappingof PDLA. Using control values of pH_(e)=6.77 (extracellular pH) and pHi=7.17 (intracellular pH), assuming no active transport, the effects ofintracellular ion trapping of PDLA with a pKa 3.86 would beapproximately (Gerweck L E, et al. Mol Cancer Ther. May 2006;5(5):1275-1279):

pH_(e)=3.86 + log [PDLA_(e) ⁻]/[PDLA_(e)] { cell wall }  pH_(i)=3.86+log[PDLA_(i) ⁻]/[PDLA_(i) ] 2.91= log [PDLA_(e) ⁻]/[PDLA_(e)] { cell wall }   3.31=log [PDLA_(i) ⁻]/[PDLA_(i) ] Antilog of equations 2042=[PDLA_(e)⁻]/[PDLA_(e)] { cell wall }  813= [PDLA_(i) ⁻]/[PDLA_(i)] [PDLA_(e)]~[PDLA_(i) ] (Assuming equilibrium across the cell wall) [PDLA_(e) ⁻]/[PDLA_(i) ⁻] = 2041/813=2.51

Thus the approximate intracellular concentration of the anion oligomerwould be 2.51× that of the extracellular concentration. The [PDLA_(p)⁻]/[PDLA_(i) ⁻]=0.68 (plasma concentration to intracellularconcentration). Lactate concentration in some aggressive cancers mayrange between 10-12.9 micromoles/gm (Walenta S, et al. Cancer Res. Feb.15, 2000; 60(4):916-921; Brizel D M, et al. Int J Radiat Oncol BiolPhys. Oct. 1 2001; 51(2):349-353).

REFERENCES

1. Warburg O W F, Negelein E. The Metabolism of Tumors in the Body. JGen Physiol. 1927; 8:519-530.

2. Seyfried T N, Shelton L M. Cancer as a metabolic disease. Nutr Metab(Lond). 2010; 7:7.

3. Vaupel P. Tumor microenvironmental physiology and its implicationsfor radiation oncology. Semin Radial Oncol. July 2004; 14(3):198-206.

4. Halestrap A P. Monocarbgoxylate transporter 1. UCSD-Nature MoleculePages. 29 Oct. 2009:1-20.

5. Gerweck L E, Vijayappa S, Kozin S. Tumor pH controls the in vivoefficacy of weak acid and base chemotherapeutics. Mol Cancer Ther. May2006; 5(5):1275-1279.

6. Brooks G A. Cell-cell and intracellular lactate shuttles. J Physiol.Dec. 1, 2009; 587(Pt 23):5591-5600.

7. Gatenby R A, Gawlinski E T, Gmitro A F, Kaylor B, Gillies R J.Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res. May15, 2006; 66(10):5216-5223.

8. Chen J L, Lucas J E, Schroeder T, et al. The genomic analysis oflactic acidosis and acidosis response in human cancers. PLoS Genet.December 2008; 4(12):e1000293.

9. Madhok B M, Yeluri S, Perry S L, Hughes T A, Jayne D G.Dichloroacetate induces apoptosis and cell-cycle arrest in colorectalcancer cells. Br J Cancer. Jun. 8 2010; 102(12):1746-1752.

10. Papandreou I, Goliasova T, Denko N C. Anti-cancer drugs that targetmetabolism, is dichloroacetate the new paradigm? Int J Cancer. Oct. 18,2010.

11. Michelakis E D, Sutendra G, Dromparis P, et al. Metabolic modulationof glioblastoma with dichloroacetate. Sci Transl Med. May 12, 2010;2(31):31ra34.

12. Dwarakanath B S. Cytotoxicity, radiosensitization, andchemosensitization of tumor cells by 2-deoxy-D-glucose in vitro. JCancer Res Ther. September 2009; 5 Suppl 1:S27-31.

13. Shrivastava V, Mishra A K, Dwarakanath B S, Ravindranath T.Enhancement of radionuclide induced cytotoxicity by 2-deoxy-D-glucose inhuman tumor cell lines. J Cancer Res Ther. April-June 2006; 2(2):57-64.

14. Raiser M, Wamelink M M, Struys E A, et al. A catabolic block doesnot sufficiently explain how 2-deoxy-D-glucose inhibits cell growth.Proc Natl Acad Sci USA. Nov. 18, 2008; 105(46):17807-17811.

15. Pelicano H, Martin D S, Xu R H, Huang P. Glycolysis inhibition foranticancer treatment. Oncogene. Aug. 7, 2006; 25(34):4633-4646.

16. Prasanna V K, Venkataramana N K, Dwarakanath B S, Santhosh V.Differential responses of tumors and normal brain to the combinedtreatment of 2-DG and radiation in glioablastoma. J. Cancer Res Ther.September 2009; 5 Suppl 1:S44-47.

17. Gasior M, Yankura J, Hartman A L, French A, Rogawski M A.Anticonvulsant and proconvulsant actions of 2-deoxy-D-glucose.Epilepsia. August 2010; 51(8):1385-1394.

18. Evans W K, Shepherd F A, Mullis B. Phase II evaluation of Lonidaminein patients with advanced malignancy. Oncology. 1984; 41 Suppl 1:69-77.

19. Gatenby R A, Gillies R J. Glycolysis in cancer: a potential targetfor therapy. Int J Biochem Cell Biol. 2007; 39(7-8):1358-1366.

20. Ikada Y, Jamshidi, K., Tsuji, H. Hyon, S.-H. Macromolecules. 1987;20:904.

21. Tsuji H. Poly(lactide) stereocomplexes: formation, structure,properties, degradation, and applications. Macromol Biosci. Jul. 14,2005; 5(7):569-597.

22. deJong S J, van Dijik-Wolthuis, W. N. E., Kettenes-van den Bosch, J.J., et al. Monodisperse enantiomeric lactic acid oligomers: Preparation,characterization, and stereocomplex formation. Macromolecules. 1998;31(19):6397-6402.

23. Hennink W E, De Jong S J, Bos G W, Veldhuis T F, van Nostrum C F.Biodegradable dextran hydrogels crosslinked by stereocomplex formationfor the controlled release of pharmaceutical proteins. Int J Pharm. Jun.11, 2004; 277(1-2):99-104.

24. Slager J, Domb A J. Biopolymer stereocomplexes. Adv Drug Deliv Rev.Apr. 25 2003; 55(4):549-583.

25. Slager J, Domb A J. Stereocomplexes based on poly(lactic acid) andinsulin: formulation and release studies. Biomaterials. November 2002;23(22):4389-4396.

26. Pandey A, Aswath P B. Microwave synthesis of poly(L-lactic acid). JBiomater Sci Polym Ed. 2009; 20(1):33-48.

27. Levesque M C, Misukonis M A, O'Loughlin C W, et al. IL-4 andinterferon gamma regulate expression of inducible nitric oxide synthasein chronic lymphocytic leukemia cells. Leukemia. February 2003;17(2):442-450.

28. Walenta S, Wetterling M, Lehrke M, et al. High lactate levelspredict likelihood of metastases, tumor recurrence, and restrictedpatient survival in human cervical cancers. Cancer Res. Feb. 15, 2000;60(4):916-921.

29. Brizel D M, Schroeder T, Scher R L, et al. Elevated tumor lactateconcentrations predict for an increased risk of metastases inhead-and-neck cancer. Int J Radiat Oncol Biol Phys. Oct. 1, 2001;51(2):349-353.

30. Lipinski C A, Lombardo F, Dominy B W, Feeney P J. Experimental andcomputational approaches to estimate solubility and permeability in drugdiscovery and development settings. Adv Drug Deliv Rev, Mar. 1, 2001;46(1-3):3-26.

31. Schwickert G, Walenta S, Sundfor K, Rofstad E K, Mueller-Klieser W.Correlation of high lactate levels in human cervical cancer withincidence of metastasis. Cancer Res. Nov. 1, 1995; 55(21):4757-4759.

32. Walenta S, Salameh A, Lyng H, et al. Correlation of high lactatelevels in head and neck tumors with incidence of metastasis. Am JPathol. February 1997; 150(2):409-415.

33. Van Tomme S R, Hennink W E. Biodegradable dextran hydrogels forprotein delivery applications. Expert Rev Med Devices. March 2007;4(2):147-164.

34. Van Tomme S R., Mens A, van Nostrum C F, Hennink W E. Macroscopichydrogels by self-assembly of oligolactate-grafted dextran microspheres.Biomacromolecules. January 2008; 9(1):158-165.

35. Walenta S, Schroeder T, Mueller-Klieser W. Metabolic mapping withbioluminescence: basic and clinical relevance. Biomol Eng. February2002; 18(6):249-262.

36. Yamagata M, Hasuda K, Stamato T, Tannock I F. The contribution oflactic acid to acidification of tumours: studies of variant cellslacking lactate dehydrogenase. Br J Cancer. June 1998; 77(11):1726-1731.

37. Kennedy K M, Dewhirst M W. Tumor metabolism of lactate: theinfluence and therapeutic potential for MCT and CD 147 regulation.Future Oncol. January 2010; 6(1):127-148.

38. Izumi H, Torigoe T, Ishiguchi H, et al. Cellular pH regulators:potentially promising molecular targets for cancer chemotherapy. CancerTreat Rev. December 2003; 29(6):541-549.

39. Joukyuu R, Mizuno S, Amakawa T, Tsukada T, Nishina T, Kitamura M.Hereditary complete deficiency of lactate dehydrogenase H-subunit. ClinChem. April 1989; 35(4):687-690.

40. Kanno T, Sudo K, Takeuchi I, et al. Hereditary deficiency of lactatedehydrogenase M-subunit. Clin Chim Acta. Dec. 8, 1980; 108(2):267-276.

41. Wakabayashi H, Tsuchiya M, Yoshino K, Kaku K, Shigei H. Hereditarydeficiency of lactate dehydrogenase H-subunit. Intern Med. July 1996;35(7):550-554.

42. Tsuji H, Miyauchi S. Enzymatic hydrolysis of poly(lactide)s: effectsof molecular weight, L-lactide content, and enantiomeric anddiastereoisomeric polymer blending. Biomacromolecules. Summer 2001;2(2):597-604.

43. Tsuji H. In vitro hydrolysis of blends from enantiomericpoly(lactide)s. Part 4: well-homo-crystallized blend and nonblendedfilms. Biomaterials. February 2003; 24(4):537-547.

44. Tsuji H, Del Carpio C A. In vitro hydrolysis of blends fromenantiomeric poly(lactide)s. 3. Homocrystallized and amorphous blendfilms. Biomacromolecules. January-February 2003; 4(1):7-11.

45. Tsuji H, Ikarashi K. In vitro hydrolysis of poly(L-lactide)crystalline residues as extended-chain crystallites. Part I: long-termhydrolysis in phosphate-buffered solution at 37 degrees C. Biomaterials.November 2004; 25(24):5449-5455.

46. Bos G W, Hennink W E, Brouwer L A, et al. Tissue reactions of insitu formed dextran hydrogels crosslinked by stereocomplex formationafter subcutaneous implantation in rats. Biomaterials. June 2005;26(18):3901-3909.

47. Slager J, Domb A J. Hetero-stereocomplexes of D-poly(lactic acid)and the LHRH analogue leuprolide. Application in controlled release. EurJ Pharm Biopharm, November 2004; 58(3):461-469.

48. Stenekes R J, Loebis A E, Fernandes C M, Crommelin D J, Hennink W E.Controlled release of liposomes from biodegradable dextran microspheres:a novel delivery concept. Pharm Res. June 2000; 17(6):690-695.

49. Panyam J, Labhasetwar V. Targeting intracellular targets. Curr DrugDeliv. July 2004; 1(3):235-247.

50. Petersen C. D-lactic acidosis. Nutr Clin Pract. December 2005;20(6):634-645.

51. Uribarri J, Oh M S, Carroll H J. D-lactic acidosis. A review ofclinical presentation, biochemical features, and pathophysiologicmechanisms. Medicine (Baltimore). March 1998; 77(2):73-82.

1-13. (canceled)
 14. A composition comprising isolated PDLA or anequivalent, derivative or analog thereof having a molecular weight (MW)of less than 500 Daltons, wherein the composition is substantially freeof PDLA having a MW greater than 500 Daltons.
 15. A compositioncomprising isolated PDLA or an equivalent, derivative or analog thereofhaving fewer than 12 monomers of D-lactic acid, wherein the compositionis substantially free of PDLA having more than 12 monomers of D-lacticacid.
 16. The composition of claim 14, wherein the PDLA or anequivalent, derivative or analog thereof has fewer than 7 monomers ofD-lactic acid.
 17. The composition of claim 16, wherein the compositionis substantially free of PDLA or an equivalent, derivative or analogthereof having more than 7 monomers of D-lactic acid.
 18. Thecomposition of claim 16, wherein the PDLA or an equivalent, derivative,or analog thereof has between 2 to 4 monomers of D-lactic acid.
 19. Thecomposition of claim 18, wherein the composition is substantially freeof PDLA or an equivalent, derivative or analog thereof having fewer than2 monomers of D-lactic acid.
 20. The composition of claim 18, whereinthe composition is substantially free of isolated PDLA (PDLA) or anequivalent, derivative or analog thereof having more than 4 monomers ofD-lactic acid.
 21. A kit for sequestering, depleting, measuring, ordetecting L-lactic acid in solution, in cell culture, in serum, in acell, or in an animal comprising PDLA or an equivalent, derivative oranalog thereof either unmodified or modified. 22-40. (canceled)
 41. Thecomposition of claim 15, wherein the PDLA or an equivalent, derivativeor analog thereof has fewer than 7 monomers of D-lactic acid.